Expression of Sirtuin 1 and 2 Is Associated with Poor Prognosis in Non-Small Cell Lung Cancer Patients

Sirtuin 1 (SIRT1) and sirtuin 2 (SIRT2) are NAD+-dependent protein deacetylases involved in the regulation of key cancer-associated genes. In this study we evaluated the relevance of these deacetylases in lung cancer biology

Combination of high levels of SIRT1 and SIRT2 proteins predicts shorter RFS and OS.

<p>Kaplan-Meier curves of RFS (A, C, E) and OS (B, D, F) for SIRT1 (A, B), SIRT2 (C, D) and the combination of SIRT1 and SIRT2 (E, F) as assessed by immunohistochemical staining.</p

Tnv-1 regulates TP53, SIRT1 and CDKN1A expression levels in TP53 wild-type NSCLC cells.

<p>A, TP53 wild-type A549 and H460 cells were treated with 10 μM tnv-1 for three days (3 d) and five days (5 d). Levels of SIRT1 and TP53 were determined by Western blot analysis using β-actin as loading control. B and C, NSCLC cells were treated with 10 μM tnv-1 for 72 hours. Sirtuin 1 (B) and p21 (CDKN1A) mRNA expression (C) was determined by real-time PCR and normalized to IPO8 mRNA levels. Error bars, SD.</p

Treatment with tnv-1 decreases growth of NSCLC cell lines.

<p>A, NSCLC cell lines were treated with 10 μM tnv-1. After three (3 d) and five (5 d) days the proliferation was measured by MTT assay. Relative proliferation values were obtained by dividing the absorbance values with those of the DMSO controls. Error bars represent standard deviation (SD). **, P < 0.01. B, Representative image of clonogenic assays (anchorage dependent growth). Cells were seeded and treated with tnv-1 (10 μM). After 10 days, colony formation was determined. C, Quantification of soft agar colony formation (anchorage independent growth) after tnv-1 treatment (10 μM) of NSCLC cell lines. Mean relative colony numbers and SD are shown. **, P < 0.01. D, Cell cycle distribution of NSCLC cells. Cells were treated with tnv-1 (10 μM) for 72 h, collected and analyzed by flow cytometry. E, Effect of tnv-1 (10 μM) on apoptosis. Cells were treated (48 h), collected and the cell death was assessed by Annexin V labeling. Error bars, SD.</p

Clinicopathological features of 105 NSCLC patients.

<p>ADC, adenocarcinomas; SCC, squamous cell carcinoma; WD, well differentiated; MD, moderately differentiated; PD, poorly differentiated; SD, standard deviation.</p><p>Clinicopathological features of 105 NSCLC patients.</p

SIRT1 and SIRT2 are upregulated in NSCLC.

<p>(A, B) Immunoblotting of SIRT1 (A) and SIRT2 (B) in NSCLC cell lines and normal immortalized lung epithelial HBEC-3KT cells. The figure is representative of three different experiments. β-actin was used as control. (C-H) Representative immunohistochemical staining for SIRT1 (C, D, G) and SIRT2 (E, F, H) in normal human lung (C-F) and tumor (G, H) specimens expressing different levels of SIRT1 and SIRT2 proteins. In tumors, SIRT1 expression is predominantly nuclear whereas SIRT2 immunoreactivity is mostly localized in the cytoplasm. Bar = 50 μm.</p

Downregulation of SIRT1 and SIRT2 inhibits proliferation of NSCLC cells.

<p>A, immunoblotting of SIRT1, SIRT2 and β-actin in A549 and H1299 cells transfected with scrambled siRNA (scr), siRNA targeting SIRT1 (siSIRT1 #1 or siSIRT1 #2) or SIRT2 (siSIRT2 #1 and siSIRT2 #2). The inhibition was verified after 72 h by Western blotting. B, A549 and H1299 cells were transfected with different siRNAs as indicated. MTT assay was performed 72 h post-transfection. The figure is representative of three different experiments: *, P < 0.05; **, P < 0.01.</p